Application of thin-layer chromatography image analysis technique in quantitative determination of sphingomyelin

A sensitive and convenient method for sphingomyelin determination was developed based on thinlayer chromatography (TLC) and image-processing analysis. The mobile phase composition, detection and quantification conditions were systematically investigated through several trials. The molybdenum blue reaction allowed specific detection of the phospholipid with a high sensitivity and a wide linear range. Digital images of TLC plate chromatograms were captured with flatbed scanner and converted into peak chromatograms using TLSee® software and quantitative analysis was conducted. The linearity, sensitivity, accuracy and precision of the system were evaluated. The limits of detection and quantification were 0.5 and 1.7 μg/spot, respectively. Separation of the mixture consisting of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine was also carried out.     

Identification, separation, and quantitative analysis of derivatives of nucleosides and nucleotides by thin-layer chromatography on cellulose

Summary The thin-layer chromatography of nucleoside and nucleotide derivatives on cellulose has been carried out, and R _f values and conditions for the separation and quantitative analysis of these mixtures by this method have been given.Khimiya Frirodnykh Soedinenii, Vol. 1, No. 5, pp. 335–342, 1965     

Uncertainty in quantitative thin-layer chromatography

Globalization forces analysts to demand extended control of variability in analytical measurements. A calculation procedure named the "error budget model" following recommendations proposed more than 20 years ago by the Bureau International des Poids et Mesures is established as a rule for evaluating and expressing the measurement uncertainty across a broad spectrum of measurements. This metrological approach common in physical measurement is not applicable in separation techniques and cannot quantitate measurement uncertainty. Our experiments show that it can be used as a planning tool in the validation of thin-layer chromatographic (TLC) methods. A computer program that quantitates uncertainty components associated with potential sources of uncertainty in quantitative TLC is prepared and tested with experimental data. TLC plates with different qualities of stationary phases (TLC and high-performance TLC) spotted with different types of samples are measured. Application is performed manually and automatically. Plates are scanned with UV-vis scanners and a video documentation system in remission and transmission mode and fluorescence. Although the calculated values are close to the values obtained with validation procedures, the error budget approach cannot substitute validation. Calculated results can predict critical points in real quantitative TLC, but they cannot confirm the validity of a selected chromatographic procedure.     

The application of thin-layer and gas chromatography to separation and identification of carbofuran and its metabolites

Conditions of chromatographical separation and identification by means of TLC and GLC were established for carbofuran and its selected metabolites, and some linear correlations

were found for the R_M and log t_R coefficient. It was stressed that additivity of chromatographic coefficients in the case of the examined substances could help with their qualitative identification when a complete set of standard compounds is not available.     

Alcohol separation by thin-layer chromatography. II

Summary Thin-layer chromatography on rice starch, maize starch, cellulose and talc makes possible the separation of the homologous series of 3,5-dinitrobenzoates of aliphatic alcohols C₁ to C₂₀. Six different solvent systems have been employed. Dependence of R _f-values on the number of C-atoms on various supports is discussed.Part I: Fresenius Z. Anal. Chem. 264, 415 (1973)     

Improved separation of amino acids by thin-layer chromatography

Summary The separation of 35 amino acids on Avicel F layers was investigated and 6 solvent systems are recommended for use either singly or in combination in 2-dimensional chromatography. The mechanisms and limitations of these methods are discussed.     

Class separation of bile lipids by thin-layer chromatography

A thin-layer chromatography technique is described that permits separation of each class of bile lipid, such as cholesterol, free (unconjugated) bile acids, glycine- and taurine-conjugated bile acids and phospholipids, in a single run. The use of silica gel G-aluminium pre-coated sheets facilitates further processing, such as the extraction in situ of each class of separated bile lipids for determination by conventional methods.     

Determination of diazepam and its metabolites by high-performance liquid chromatography and thin-layer chromatography

A sensitive, simple high-performance liquid chromatographic assay, capable of simultaneously measuring diazepam, its active metabolites oxazepam, temazepam and N-desmethyldiazepam and two phenyl hydroxylated metabolites, 4'-hydroxy-N-desmethyldiazepam and 4'-hydroxydiazepam, is described. The assay is easily modified to include separation of additional metabolite(s), e.g. oxazepam glucuronide(s). A thin-layer chromatographic assay, which resolves diazepam, the active metabolites and the two phenyl hydroxylated derivatives in one solvent system, is also reported. Application of these procedures to the quantitation of diazepam and its metabolites was shown, after delivery of diazepam (5 micrograms/ml or 16 microM) at a constant flow-rate (10 ml/min per liver) through the single-pass perfused rat liver preparation. Blood perfusion medium and bile were analysed for parent drug and metabolites before and after enzyme hydrolysis. These assay methods are found to be particularly pertinent and useful in providing a more comprehensive metabolic profile of diazepam metabolism, especially when aromatic hydroxylation pathways predominate.     

Direct separation and quantitative determination of glimepiride isomers by high performance liquid chromatography

A simple and sensitive high‐performance liquid chromatographic procedure for the determination of the trans isomer of glimepiride is reported. Chromatography accomplished direct separation of the cis and trans isomers of glimepiride on a Dikmonsil C18 (250×4.6 mm, 5 μm) column with a mobile phase consisting of methanol‐acetonitrile‐NH₄Ac buffer solution (1.5 mol L^–1, pH = 4.5) (1.1 : 1.3 : 1.0, v/v) at a flow rate 0.5 mL min^–1. The resolution (R_S) was 1.73 with a retention time of 24.885 and 23.018 min for the cis and the trans isomer, respectively. A standard linear calibration curve was established for the trans isomer of glimepiride over the range of 4.95–198.00 μg mL^–1 with a correlation coefficient of 0.99997. This method has been successfully used to analyze four different kinds of glimepiride product.     

Rapid separation of lactucin and lactucopicrin from Cichorium glandulosum by medium-pressure preparative liquid chromatography and quantitative analyses by high-performance thin-layer chromatography

Medium-pressure preparative liquid chromatography (MPPLC) was used to isolate and prepare lactucin and lactucopicrin from the whole herb of Cichorium glandulosum. After extracting the methanolic extract of the whole plant with petroleum ether and ethyl acetate several times to obtain ethyl acetate extract, the crude products, namely, lactucin and lactucopicrin were separated using MPPLC and thin-layer chromatography (TLC) tracking, and their purity rates reached more than 80%. The qualitative and quantitative analyses of lactucin and lactucopicrin were carried out by high-performance thin-layer chromatography (HPTLC) on the whole herb of C. glandulosum. The contents of lactucin and lactucopicrin were determined by scanning at 256 nm in the whole herb of C. glandulosum. The R_F values of lactucin and lactucopicrin were 0.42 ± 0.05 and 0.65 ± 0.05 with linear ranges of 0.498–2.988 and 0.499–2.994 μg/band, respectively. The correlation coefficients were 0.9938 and 0.9946, respectively, thereby showing a good linear relationship. The average recoveries were 99.96% and 99.52%, and the relative standard deviations (RSDs) were 2.49% and 2.45%, respectively. The crude products, namely, lactucin and lactucopicrin, can be isolated with high purity from the whole herbs of C. glandulosum by MPPLC. The lactucin and lactucopicrin contents of C. glandulosum can be determined rapidly and accurately by HPTLC.

     

Simultaneous determination of insecticides, acaricides and fungicides by thin-layer chromatography

A method is described for the determination of residual amounts of some insecticides, acaricides and fungicides in fresh and processed fruits. Optimum conditions were established for the extraction of eighteen pesticides, purification of the extracts by column chromatography and subsequent two-dimensional thin-layer chromatography on silica gel GF254 with cyclohexane-acetone (10:1) and light petroleum-benzene-ethanol (65:30:5) as solvents and with detection under UV light at 254 and 366 nm followed by a 0.1% solution of bromophenol blue.     

Theoretical calculation of the optimum separation in thin-layer chromatography

Summary Up to now the optimum composition of a binary mobile phase for separation in thin-layer chromatography has been determined mainly by the method of successive trials. In this paper a simple way is presented to determine the mobile phase composition and the maximum difference in the R_M values of two substances. The equation introduced has been illustrated with examples of adsorption thin-layer chromatography on magnesium silicate. Calculation of the largest difference in the R_M values of two substances in relation to the composition of the binary mobile phase has been compared with the corresponding values determined experimentally. A good agreement of the obtained theoretical data and experimental values has been found.     

A rapid separation of citrus carotenoids by thin-layer chromatography

Summary A rapid separation of citrus carotenoid pigments is achieved by successive thin-layer chromatographic separations on two different adsorbents. The first chromatography on silica gel G developed with the solvent system acetone-petroleum ether (30∶70) gives a preliminary fractionation into groups with different polarity (hydrocarbons, monols, diols and polyols). A further separation of each group into individual carotenoids is obtained by rechromatography on MgO-Kieselguhr (1∶1, w/w). The same solvent system is used, the acetone percentage being increased according to the polarity of each group (from 4% to 30%).